1. Field of the Invention
This invention relates to formulations comprising biological samples preserved as glassy dry powders and hydrophobic, non-toxic carriers, the formulations being adapted for the long-term storage and delivery of the biological samples at selected ambient or higher storage temperatures, and to methods for preparing these formulations. More particularly, the invention relates to hydrophobic liquid formulations adapted for ambient or higher temperature storage and delivery of vaccines and vectors.
2. Description of the Related Art
Peptides, proteins, nucleic acids, hormones, antibodies and other biologically active molecules, viruses and bacteria, vectors, cells, and small multicellular specimens have a broad range of uses, including for example, human and veterinary pharmaceuticals, molecular biology, gene therapy, as well as in the food industries. Typically, these bioactive materials are active in aqueous environments; thus, conventional formulations of such samples have been in aqueous solutions. However, many bioactive materials are sensitive to degradation and loss of activity and/or viability in aqueous solutions, particularly at ambient or higher temperatures. Accordingly, bioactive materials often require refrigeration or have short shelf lives under ambient conditions. Further, many bioactive materials have only limited solubility in aqueous solutions. Even when they are soluble at high concentrations, they are prone to aggregation and precipitation.
Bioactive materials can degrade via a number of chemical mechanisms known in the art. Water is a reactant in nearly all of these degradation pathways. Further, water acts as a plasticizer, which allows unfolding and aggregation of proteins. Since water is a participant in almost all degradation pathways, reduction of the aqueous solution or suspension of bioactive materials to a dry powder provides an alternative formulation methodology to enhance the stability of such samples. Bioactive materials can be dried using various techniques, including freeze-drying, spray-drying and dessication. Aqueous solutions of bioactive materials are dried and stored as dry powders until their use is required.
A serious drawback to drying of bioactive materials is that often end uses of such materials require some sort of liquid form. Parenteral injection and the use of drug delivery devices for sustained delivery of drug are two examples of applications where one would like to use bioactive materials in a liquid form. For injection, dried bioactive materials must be reconstituted, adding additional steps which are time-consuming and where contamination may occur, and exposing the bioactive materials to potentially destabilizing conditions.
The sustained parenteral delivery of drugs provides many advantages. The use of implantable devices for sustained delivery of a wide variety of drugs or other beneficial agents is well known in the art. Typical devices are described, for example, in U.S. Pat. Nos. 5,034,229, 5,057,318 and 5,110,596. The disclosure of each of these patents is incorporated in its entirety herein by reference thereto.
Proteins are only marginally soluble in non-aqueous solvents, and such solvents typically unfold and denature proteins. Solubilization of native proteins in non-aqueous solvents typically requires derivatization or complexation of the protein. In attempting to achieve enzymatic catalysis in organic media, certain catalytic enzymes can be suspended in non-aqueous vehicles as powders, typically in hydrophilic organic solvents including alcohol ketones and esters. With enzyme hydration levels ≧10% and/or the addition of low molecular weight protic compounds, these enzymes can have enough conformational mobility to exhibit appreciable enzymatic activity. Optimal activity levels are apparently achieved at enzyme hydration of approximately 30%. At a minimum, such enzymatic activity requires a level of “essential water” hydrating the protein. However, hydration levels (generally 10–40% w/w water/protein) and/or protic solvents, such as those used in these studies, typically result in unacceptable stability of proteins for pharmaceutical purposes. A further requirement for catalysis in non-aqueous solvents is that the enzyme be dried from a solution having a pH near the optimal pH for the enzymatic activity. This pH limitation is detrimental to storage of protein pharmaceuticals, because most protein degradation mechanisms are pH dependent, and it is often the case that proteins are most stable when dried at pH values far from the value where they exhibit bioactivity. Further, such catalytic enzyme systems are not amenable to the addition of protein stabilizers, particularly those that function by hydrogen bonding to the protein and reducing enzyme hydration (e.g., carbohydrates).
The field of gene therapy or gene transfer is advancing both experimentally and clinically. Nucleic acids have been transferred into cells using viral vectors such as adenovirus, retrovirus, adeno-associated virus, vaccinia virus, and sindbis virus, among others. Non-viral methods have also been used, including calcium phosphate precipitation, DEAE dextran, injection of naked DNA, electroporation, cochleates, cationic lipid complexes, liposomes, polymers (such as dendrimers and PLGA), virosomes, and the like.
DNA complexed with cationic lipids and/or liposomes has been shown to be an efficient means of transfecting a variety of mammalian cells. Such complexes are simple to prepare and may be used with a wide variety of DNA's and RNA's with little restriction to the size of nucleic acid. They have the ability to transfect many different cell types with efficiency and are not immunogenic. Current nucleic acid formulations, including DNA/liposome and RNA/liposome complexes, must be mixed shortly before administration, resulting in inconvenience in manufacture, shipping, storage and administration. Frequently, these two-part formulations are not very highly concentrated, requiring the administration of large volumes of solution. Dry powder formulations containing lyophilized nucleic acid/liposome complexes have also been used, but they require reconstitution with suitable aqueous solution just prior to administration. Aqueous complexes are inherently unstable and lose most, if not all, of their transfection activity within hours or a few days.
Consequently, there is a need for formulations of viral and bacterial vaccines and vectors, pharmaceutical materials, probiotics, and other industrially important bioactive materials that can overcome these limitations of the prior art. Such formulations should maintain the stability of the active materials, preferably at both room and body temperatures (25° and 37° C.), and exist in at least a flowable state for injection, pulmonary delivery, incorporation into delivery systems designed for immediate, delayed, or long term administration or other means of administration.